Molecular evidence of Borrelia spp. in bats from Córdoba Department, northwest Colombia

Background The genus Borrelia is composed of two well-defined monophyletic groups, the Borrelia burgdorferi sensu lato complex (Bb) and the relapsing fever (RF) group borreliae. Recently, a third group, associated with reptiles and echidnas, has been described. In general, RF group borreliae use rodents as reservoir hosts; although neotropical bats may also be involved as important hosts, with scarce knowledge regarding this association. The objective of this study was to detect the presence of Borrelia spp. DNA in bats from the department of Córdoba in northwest Colombia. Methods During September 2020 and June 2021, 205 bats were captured in six municipalities of Córdoba department, Colombia. Specimens were identified using taxonomic keys and DNA was extracted from spleen samples. A Borrelia-specific real-time PCR was performed for the 16S rRNA gene. Fragments of the 16S rRNA and flaB genes were amplified in the positive samples by conventional PCR. The detected amplicons were sequenced by the Sanger method. Phylogenetic reconstruction was performed in IQ-TREE with maximum likelihood based on the substitution model TPM3+F+I+G4 with bootstrap values deduced from 1000 replicates. Results Overall, 10.2% (21/205) of the samples were found positive by qPCR; of these, 81% (17/21) and 66.6% (14/21) amplified 16S rRNA and flaB genes, respectively. qPCR-positive samples were then subjected to conventional nested and semi-nested PCR to amplify 16S rRNA and flaB gene fragments. Nine positive samples for both genes were sequenced, and seven and six sequences were of good quality for the 16S rRNA and flaB genes, respectively. The DNA of Borrelia spp. was detected in the insectivorous and fruit bats Artibeus lituratus, Carollia perspicillata, Glossophaga soricina, Phyllostomus discolor, and Uroderma sp. The 16S rRNA gene sequences showed 97.66–98.47% identity with “Borrelia sp. clone Omi3,” “Borrelia sp. RT1S,” and Borrelia sp. 2374; the closest identities for the flaB gene were 94.02–98.04% with “Borrelia sp. Macaregua.” For the 16S rRNA gene, the phylogenetic analysis showed a grouping with “Candidatus Borrelia ivorensis” and “Ca. Borrelia africana,” and for the flaB gene showed a grouping with Borrelia sp. Macaregua and Borrelia sp. Potiretama. The pathogenic role of the Borrelia detected in this study is unknown. Conclusions We describe the first molecular evidence of Borrelia spp. in the department of Córdoba, Colombia, highlighting that several bat species harbor Borrelia spirochetes. Graphical Abstract

López et al. Parasites & Vectors (2023) 16:5 Background Pathogenic species of the genus Borrelia are zoonotic bacteria that cause emerging and re-emerging infectious diseases worldwide and constitute a major threat to public health [1]. This genus is composed of two welldefined monophyletic groups: the Borrelia burgdorferi sensu lato (Bb) complex, which cause Lyme borreliosis and are transmitted by hard ticks of the genus Ixodes, and borreliae of the relapsing fever (RF) group, transmitted mainly by soft ticks of the genus Ornithodoros, some species by ixodid ticks (e.g., Borrelia miyamotoi), and Borrelia recurrentis, transmitted by the clothing louse [2]. Recently, a third group associated with reptiles and echidnas (Tachyglossus aculeatus) has been described [3,4], and share a common ancestor with the RF clade [3,4].
Given their intimate relationship with emerging microorganisms that cause serious infections in humans, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) [22], and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [23], bats have recently been the center of attention for scientists. A remarkable fact is that bats harbor more viruses than rodents [24]. However, bacteria harbored by these mammals are neglected, even though they can represent zoonotic pathogens [25]. For example, RF group spirochetes such as Ca. Borrelia fainii and Ca. Borrelia johnsonii are among those neglected agents [26,27]. In order to contribute to the understanding of Borrelia spp. associated with chiropterans, we carried out a prospective study to detect DNA of Borrelia in organs of bats derived from a COVID-19 study, collected in the department of Córdoba, Colombia.

Study area and bat captures
Field trips were carried out during September 2020 and June 2021 in six municipalities of Córdoba Department (Montelíbano, Tierralta, San Antero, Montería, Lorica, and Moñitos) in urban, peri-urban and rural areas with similar environmental conditions: altitude of 12-87 m, 25-28 °C, and average relative humidity of 81% ( Fig. 1; Table 2).
Bats were captured using mist nets (6 m × 2 m). Males and females without signs of pregnancy were included in the study. Captured bats were euthanized with an overdose of sodium pentobarbital (200 mg) at a dose of 0.05 mg/g [28]. Following taxonomic identification using dichotomous keys [29], spleen samples were extracted, stored in sterile tubes, and kept at −80 °C until processing. The capture of bats was carried out under the permits of the National Environmental Licensing Authority (ANLA), Resolution No. 00914. All the procedures were approved by the ethics committee of the Faculty of Veterinary Medicine and Zootechnics of the Universidad de Córdoba (No. 003 of December 6, 2019).

Molecular and phylogenetic analyses
DNA extraction was performed in spleen samples using the GeneJET Genomic DNA Purification Kit (Thermo Scientific) following the manufacturer's instructions. A conventional polymerase chain reaction (cPCR) targeting the mammalian ß-actin gene was performed as an internal control for each extraction [30]. Positive samples were then submitted to a real-time PCR (qPCR) specific for the genus Borrelia, [31]. Samples with cycle threshold values ≤ 33 were considered positive and then subjected to conventional nested and semi-nested PCRs to amplify 16S rRNA and flaB genes fragments [32,33]. The annealing temperature for the 16S rRNA gene in the cPCR was modified in the first round (FD3 [f ]/T50 [r]) and second round (Rec4 [f ]/Rec9 [r]) from 56 °C to 54 °C (Table 1). Genomic DNA of Borrelia anserina and moleculargrade water were used as positive and negative controls, respectively. Amplicons of the expected size were Sangersequenced at Macrogen (Seoul, Korea), sequences assembled with Geneious, and the consensuses were compared in GenBank using BLASTn [34].
The alignments were built in Clustal Omega [35]. For the 16S rRNA and the flaB gene, 70 and 81 sequences downloaded from GenBank were used, respectively [36]. Phylogenetic reconstructions were performed in IQ-TREE with the maximum likelihood method using the TPM3+F+I+G4 nucleotide substitution model with 1000 bootstraps. Trees were visualized and edited with iTOL v5 [37]. Brachyspira pilosicoli was used as root.

Results
Two hundred and five spleen samples from different bat species were processed. Amplicons of the expected size for the ß-actin gene were obtained in all samples, thus confirming successful DNA extraction. Overall, 10.2% (21/205) were positive for the Borrelia 16S rRNA gene by qPCR. In 81% (17/21) and 66,6% (14/21) positive samples, it was possible to amplify 16S rRNA and flaB genes by cPCR. Regarding bat species,   Table 2). In the 16S rRNA phylogeny, all the obtained sequences were grouped monophyletically and as a sister lineage to "Candidatus Borrelia ivorensis" and "Candidatus Borrelia africana" out of the RF or Lyme groups ( Fig. 2A). The flaB phylogenetic tree places all our sequences into a monophyletic clade together with haplotypes of "Borrelia sp. Macaregua" and Borrelia sp. Potiretama (Fig. 2B).

Discussion
To date, there are few studies that show the presence of Borrelia in bats. One of the first reports was made by Nicolle and Comte in 1905, with the finding of spirochetes in the blood of Vespertilio kuhli from Tunisia [41]. Then, in 1945, Nájera Angulo demonstrated the susceptibility of four bats species (Miniopterus schreibersii, Myotis myotis, Rhinolophus euryale, and Rhinolophus hipposideros minimus) from Spain to a "Hispanic spirochete" after inoculation of blood from infected guinea pig [42]. Later, in 2006, in the United States, through serological tests it was possible to verify the circulation of borreliae in Eptesicus fuscus bats [17]. Subsequently, in 2009, in England, spirochetes were identified in liver tissue stained with Warthin-Starry in a bat of the genus Pipistrellus, and it was confirmed as an RF group Borrelia, obtaining a 776-base-pair (bp) segment of the flaB gene [7].
In recent years, Ca. Borrelia fainii was reported in Myotis sp. [9], Rhinolophus pusillus, and Myotis davidii bats in China [12]. Regarding Latin America, in Mexico, Colunga-Salas et al. detected two new lineages of Borrelia, one RF and one Bb, in Saccopteryx bilineata, Choeroniscus godmani, Sturnira parvidens, and Lasiurus cinereus [11]. In Colombia, in 1968, Marinkelle and Grose observed spirochetes in a blood smear of Natalus tumidirostris [43] from the Macaregua cave, and in 2020 Muñoz-Leal et al. detected a new putative taxon within the genus Borrelia in Carollia perspicillata captured in the same cave [10]. Likewise, in 2022, Jorge et al. detected Borrelia sp. Potiretama in Desmodus rotundus bats, in the municipality of Potiretama in Brazil [21]. In the current study, we detected Borrelia DNA in five species of bats (C. perspicillata, Phyllostomus discolor, Artibeus lituratus, Glossophaga soricina, and Uroderma sp.) captured in Colombia, reinforcing the fact that bats do harbor Borrelia spirochetes.
In our study, two phylogenetic analyses were performed including Borrelia spp. previously detected in bats from South America, such as Borrelia sp. Macaregua and Borrelia sp. Potiretama [10,21]. Unfortunately, 16S rRNA sequences for Borrelia sp. Macaregua are not available in GenBank, so it was not possible to perform comparisons with this species using this gene. Interestingly, 16S rRNA phylogeny depicts the group of Borrelia spp. detected in this study as paraphyletic regarding Borrelia sp. Potiretama reported in bats from Brazil, and Ca. Borrelia ivorensis and Ca. Borrelia africana, two species detected in African ticks, as closely related. The fact that some South American borreliae are phylogenetically closer to their African representatives has also been observed for spirochetes characterized in soft ticks from Brazil and Chile [38,44]. However, any conclusion regarding the phylogenetic position of the Borrelia spp. detected in our study is premature, since we obtained short 16S rRNA gene sequences. Indeed, the fact that the flaB tree showed the Borrelia sequences of our study forming a monophyletic group with Borrelia sp. Potiretama, contradicting the topology of the 16S rRNA tree, could also be explained by the limited data submitted to analysis for this gene (381-435 bp).
A monophyletic group of Borrelia spp. associated with neotropical bats roosting in caves has been recently proposed based primarily on phylogenetic analyses of flaB sequences [10,21]. Our study supports this hypothesis, adding more genovariants to this group of bat species inhabiting wooded areas. Because of their defense-immune tolerance capacities, bats are considered excellent reservoirs that favor the emergence of novel viruses [45]. The reasons that South American bats seem to harbor such a remarkable diversity of Borrelia haplotypes could relate to their immunological system as well. Finally, isolating Borrelia spp. circulating in bats and sequencing their genomes should now be the focus in order to clearly elucidate the phylogenetic relationships. The study of Borrelia in bats is important because several species associated with these mammals, such as Ca. Borrelia fainii and Ca. Borrelia johnsonii, have recently been detected in humans in Africa and the United States, respectively [9,12,26,27]. Therefore, the pathogenic roles of spirochetes detected in neotropical bats should be further investigated.

Bb
Borrelia burgdorferi sensu lato RF Borreliae of the relapsing fever group